Quantification of algal biofilms colonising building materials: chlorophyll a measured by PAM-fluorometry as a biomass parameter

The aim of this study was to quantify algal colonisation on anthropogenic surfaces (viz. building facades and roof tiles) using chlorophyll a (chl a) as a specific biomarker. Chl a was estimated as the initial fluorescence F0 of 'dark adapted' algae using a pulse-modulated fluorometer (PAM-2000). Four isolates of aeroterrestrial green algae and one aquatic isolate were included in this study. The chl a concentration and F0 showed an exponential relationship in the tested range between 0 and 400 mg chl a m(-2). The relationship was linear at chl a concentrations <20 mg m(-2). Exponential and linear models are presented for the single isolates with large coefficients of determination (exponential: r2 > 0.94, linear: r2 > 0.92). The specific power of this fluorometric method is the detection of initial algal colonisation on surfaces in thin or young biofilms down to 3.5 mg chl a m(-2), which corresponds to an abundances of the investigated isolates between 0.2 and 1.5 million cells cm(-2).     

Estimation of chlorophyll content and daily primary production of the major algal groups by means of multiwavelength-excitation PAM chlorophyll fluorometry: performance and methodological limits

The performance and methodological limits of the Phyto-PAM chlorophyll fluorometer were investigated with laboratory grown algae cultures and natural phytoplankton from the rivers Saar and Saale. The Phyto-PAM is a 4-wavelength chlorophyll fluorometer with the functional combination of chlorophyll (Chl) estimation and assessment of photosynthetic activity, both differentiated into the main algal groups. The reliability of fluorescence-based Chl estimation strongly depends on the group specific calibration of the instrument and the resulting chlorophyll/fluorescence (Chl/F) ratios in reference algal cultures. A very high reliability of the Chl estimation was obtained in the case of constant Chl/F-ratios. Algae grown at different light intensities showed marked differences in Chl/F-ratios, reflecting differences in pigment composition and Chl a specific absorption (a*). When the Phyto-PAM was calibrated with laboratory grown diatoms, the Chl a in river grown diatoms was underestimated, due a lower content of accessory pigments and stronger pigment packaging. While this aspect presently limits the application of PAM fluorometry in limnology, this limitation may be overcome by future technical progress in the detection of dynamic changes in Chl/F-ratio via fluorescence-based measurements of the functional PS II absorption cross-section. Practically identical Chl/F-ratios were found for the diatom-dominated waters of the rivers␣Saar and Saale, suggesting that the same instrument calibration parameters may be applied for hydrographically similar surface waters. For this particular case, despite of the present methodological limitations, the potential of PAM fluorometry in limnology could be demonstrated. Light response curves were measured to estimate primary production with a spectrally resolved model in daily courses at two sampling sites. Fluorescence based primary production was closely correlated with measured oxygen evolution rates until midday. In the afternoon, at the water surface the fluorescence approach gave higher␣rates than the measured oxygen evolution. Possible explanations for the observed differences are discussed.     

生物量精确估算模型与参数辨识方法及应用

从生物量模型的构建与参数辨识方法的改进对生物量进行精确估算。用Chebyshev多项式系的组合构建了p维连续函数空间的一组乘积型基,进而建立了生物量估算统一模型,它具有如下特点:(1)可以克服常用生物量估算模型的经验性、不稳定性、不通用性及对生物量影响因素适应性差的特点,(2)它适合于影响生物量的任何因素,故适应范围非常广且很稳定,(3)可根据实际需要及估算精度确定影响生物量的因素及其阶数大小,(4)模型对生物量的估算相当于在区间[-1,1]上进行的数值插值,变量阶数越高,所插入的点就越多,估算结果越符合实际,整个估算的插值过程与树木的树干解析与树木生长原理是相一致的。对所建模型的参数辨识方法做了探讨,经典最小二乘算法是生物量估算的最常用参数辨识方法,由于它本身固有的一些缺陷使常用最小二乘的估算精度与使用范围受到很大的限制,现代多元统计分析的偏最小二乘算法可以克服常用最小二乘的缺陷,但在提取成分时仍具有不足,针对偏最小二乘的缺陷本文对它做了改进,改进算法即能克服偏最小二乘的不足还能使估算精度大大提高。用2个案例对3种生物量估算方法做了对比分析,结果表明生物量估算统一模型与偏最小二乘改进算法精度最高,其生物量估计误差在零附近排成一条直线。     

Determination of microphytobenthic biomass using pulse-amplitude modulated minimum fluorescence

Microphytobenthos are the major intertidal primary producers in many estuarine systems and assessment of their biomass is essential in ecological studies. Algal biomass can be assessed by the use of proxy measurements, e.g. chlorophyll a (Chl a), using a lengthy and destructive extraction procedure. Fluorescence is a non-destructive and in situ alternative, recently applied to microphytobenthos, using the minimum Fluorescence signal (Fo) as a measurement of Chl a. In this study, Fo after 15 min of dark adaptation (Fo ¹⁵) showed a significant positive correlation with Chl a (r?=?0.84, p?n?=?24). The relationship between Fo ¹⁵ and Chl a extracted from sediment samples of increasing sample depth (0.2?2?mm) became less significant as sediment depth increased. The strongest relationship between Fo ¹⁵ and Chl a, expressed as concentration (mg?m^?2) and content (mg?kg^?1), was found when all data were combined, but nearly comparable levels of association were found for in situ measurements using sediment cores up to 2.0?mm deep. Results suggested that full dark adaptation was not always achieved because a significant negative correlation was observed between Fv/Fm and ambient irradiance for seasonal data. In addition, Fo ¹⁵ showed a more significant correlation with Chl a than with Fm ¹⁵, suggesting some residual level of non-photochemical quenching after 15 min in the dark. Despite the incomplete dark adaptation, the highly significant relationships between Fo ¹⁵ and both Chl a concentration and content suggest that the use of Fo ¹⁵ is a suitable in situ proxy method of estimating algal biomass.     

Estimation of soil algal biomass in salinized irrigation land: a comparison of culture dilution and chlorophyll a extraction methods

Two representative methods for quantitative estimation of soil algae, the culture dilution method and chlorophyll a extraction, were compared using soil samples collected from irrigation land in the flood plain of the River Ili, Kazakhstan, where the distribution of soil algae had been studied in the previous year. The estimate by the culture dilution method was almost the same as in the previous year, except for one site, which was enclosed by shrubs of sakusaul, Haloxylon aphyllum (Minkw.) Iljin. The important role of wind in transport of airborne algal cells was pointed out. There was a significant correlation (p<0.001) between the logarithm of the number of colonies by the culture dilution method andthe logarithm of the concentration of chlorophyll a, when data from all samples were analyzed. However, no significant correlation was observed with the data of cropland sites alone. Furthermore, the seasonal variations of both values at each site did not necessarily agree with each other. One reason for the inconsistency may have been the over estimation of chlorophyll a caused by inclusion of litter from vascular plants. Other reasons may relate to differences between the methodologies. The density of soil algae estimated by culture dilution reflects the algal biomass in a certain previous period of time. Therefore, it is suggested that the method is suitable for spatial, but not for seasonal studies. This revised version was published online in August 2006 with corrections to the Cover Date.     

Long-term monitoring of the biomass and production of lipids by Nitzschia palea for biodiesel production

Pennate diatom Nitzschia palea can be cultured in outdoor vertical-bed photobioreactors to produce biodiesel. To assess the production of biomass and lipids, non-axenic cultures of Nitzschia palea were grown outdoors, and the growth of these cultures was measured biweekly. During the annual cycle of algal culture, the culture temperature ranged from 17.3 °C to 33.5 °C, the dry weight biomass ranged from 0.11 g l⁻¹ to 0.25 g l⁻¹, light energy] ranged from 1.94 Wm⁻² to 3.9 Wm⁻² and intracellular lipid content ranged from 7.1% to 11.4% of biomass weight after drying at 60 °C. Gas chromatography/mass spectroscopy (GC/MS) analysis of n-hexane extracts showed that the intracellular lipids were primarily C14:0 myristic acid (9.01%), C15:0 pentadecyclic acid (8.26%) and two types of C16:0, palmitic acid (41.13%) and palmitoleic acid (29.25%). Gel permeation analysis showed that carboxylic acids comprised 28.9% of lipids, 16.3% of monoglycerides, 27.3% of diglycerides and 24.3% of triglycerides. Alcoholysis of lipids resulted in the conversion of about 93.9% of fatty acids to equivalent fatty acid methyl esters (FAME) or biodiesel, which, on basis of wt%, consisted primarily of C15:0 methyl myristate (8.3%), C16:0 methyl pentadecanoate] (7.2%), C17:1methyl palmitoleate (28.7%) and methyl palimtate](39.8%).     

The photosynthesis of individual algal cells during the cell cycle of Scenedesmus quadricauda studied by chlorophyll fluorescence kinetic microscopy

A microscope for imaging of chlorophyll fluorescence kinetics was equipped with a chamber that allows the growth of an immobilised population of algae and their study under well-defined conditions. Single cells of the chlorococcal alga Scenedesmus quadricauda were grown and recorded for periods of whole cell cycles (up to 48 h) displaying a normal course of cell development. Heterogeneity in fluorescence yield among individual coenobia in the population and among different cells in one coenobium were analysed. Differences were observed both in the shape of Kautsky transients and in the modulation of fluorescence parameter values during the progress of the cell cycle. The extent of heterogeneity in fluorescence parameters was cell cycle dependent – in some phases of the cycle, the population was almost homogeneous, while distinct heterogeneity was observed, in particular between the protoplast division and the release of the daughter coenobia. The heterogeneity was not random but reflected developmental processes.     

应用叶绿素荧光法测定微藻生物量的方法

为了探求叶绿素荧光值与海洋微藻生物量的关系,利用TD-700型叶绿素荧光仪测定了6种典型海洋赤潮藻在不同生长期(指数生长期和稳定生长期)时的叶绿素荧光值,同时用传统方法测定细胞密度,且利用Leica Qwin软件测量细胞大小。结果显示:微藻在同一生长时期细胞密度与叶绿素荧光值存在极显著的正相关关系,不同生长时期细胞密度也与单位叶绿素荧光值存在较显著正相关关系。因此,利用叶绿素荧光仪测定微藻生物量的方法是切实可行的,具有快捷方便、灵敏度高、可靠性强的优点。     

The light-harvesting chlorophyll a/b protein acts as a torque aligning chloroplasts in a magnetic field

Displacement of particles from the purified light-harvesting chlorophyll a/b protein aggregate (LHC) was studied in magnetic fields of various strengths (0 to 1.6 T) by polarized fluorescence measurements. Macromolecular aggregates of LHC have a considerable magnetic susceptibility which enables the particles to rotate and align with their nematic axes parallel with H. As LHC is embedded in a transmembrane direction thylakoids should align perpendicular to H, the mode of alignment experimentally observed in thylakoids. The value of the magnetic susceptibility could be estimated by relating it to the integral susceptibility of the chlorophyll molecules in LHC. The fitting of this value with the field strength dependency of the fluorescence polarization ratio (FP) revealed a relationship between the LHC content of various photosynthetic membranes and their capacity for alignment, which suggested that LHC might be the torque ordering chloroplasts in a magnetic field.Abbreviations LHC light-harvesting chlorophyll a/b protein - FP fluorescence polarization ratio, Iz/Iy     

A combination of assays reveals biomass differences in biofilms formed by Escherichia coli mutants

Aims:  The aim of this study was to develop an assay system that can quantify the amount of biomass in biofilms formed by different isogenic mutants of an Escherichia coli K-12 strain.
Methods and Results:  The reported assay, which is based on the BacTiter-Glo™ assay from Promega, uses bioluminescence to detect the intracellular concentration of ATP, which correlates with viable bacterial cell numbers. The quantitative data obtained with this ATP assay were compared to those obtained with the conventional crystal violet assay. As a qualitative control, scanning electron microscopy was performed.
Conclusions:  The ATP assay, the crystal violet assay and scanning electron microscopy yielded similar results for six of the eight strains tested. For the remaining two strains, the images from the scanning electron microscopy confirmed the results from the ATP assay.
Significance and Impact of the Study:  The ATP assay, in combination with other quantitative and qualitative assays, will allow us to perform genetic studies on the regulatory network that underlies the early steps in E. coli biofilm formation.     

Temperature-induced alterations of in vivo chlorophyll a fluorescence induction in cucumber as affected by DCMU

Induction of chlorophyll a fluorescence and photosynthesis as affected by temperature were measured in cucumber leaf discs. Abrupt changes of the maximal variable fluorescence, Fv(p), and photosynthesis were observed around 9° and 21°C when the temperature was decreased from 30° to 0°C. The temperature-dependent maximal fluorescence of DCMU-treated leaf discs showed a single change around 21°C. Temperature-induced chlorophyll a fluorescence alterations are discussed in relation to electron transport activity of the two photosystems and photosynthetic activity of the cucumber leaf discs.Abbreviations DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - Fm maximal fluorescence - Fv(p) maximal variable fluorescence - qE energy-dependent fluorescence quenching - qQ Qa-dependent fluorescence quenching     

Separation of components of the biomass from high rate algal ponds using PercollR density gradient centrifugation

The biomass of a High Rate Algal Pond was separated into individual components of algae, bacteria and detritus. The two stage technique involved mechanical and chemical disaggregation of concentrated pond samples, followed by separation on preformed Percoll^R/sucrose density gradients. Throughout a diurnal cycle, monitored in September 1990, between 85 and 90% of the total chlorophylla was recovered in the algal fraction. The greatest loss of chlorophyll from the sample occurred during the concentration stage; no further losses were encountered during physical and chemical disaggregation. The technique enabled the direct gravimetric determination of the separated algal biomass. The potential applications of the technique are discussed.     

Drought-induced changes in chlorophyll fluorescence,photosynthetic pigments,and thylakoid membrane proteins of Vigna radiata

The effects of drought on chlorophyll fluorescence characteristics of PSII, photosynthetic pigments, thylakoid membrane protein (D1), and proline content in different varieties of mung bean plants were studied. Drought stress inhibits PSII activity and induces alterations in D1 protein. We observed a greater decline in the effective quantum yield of PSII, electron transport rate, and saturating photosynthetically active photon flux density (PPFD_sat) under drought stress in var. Anand than var. K-851 and var. RMG 268. This may possibly be due to either downregulation of photosynthesis or photoinhibition process. Withholding irrigation resulted in gradual diminution in total Chl content at Day 4 of stress. HPLC analysis revealed that the quantity of β-carotene in stressed plants was always higher reaching maxima at Day 4. Photoinactivation of PSII in var. Anand includes the loss of the D1 protein, probably from greater photosynthetic damage caused by drought stress than the other two varieties.     

Potential use of lake cyanobacterial biomass as fertilizer for the cultivation of food crops

This study points to the utility of Aulosira fertilissima and algal biomass of the Upper and Lower lake of Bhopal as a cheap and effective green manure for rice and mustard.     

Effects of anaerobiosis as probed by the polyphasic chlorophyll a fluorescence rise kinetic in pea (Pisum sativum L.)

We analysed the changes of the chlorophyll (Chl)a fluorescence rise kinetic (from 50 s to 1 s) that occur when leaves or chloroplasts of pea ( Pisum sativum L.) are incubated under anaerobic conditions in the dark. In control leaves, Chl a fluorescence followed a typical O-J-I-P polyphasic rise [Strasser et al. (1995) Photochem Photobiol 61: 32–42]. Anaerobiosis modified the shape of the transient with the main effect being a time-dependent increase in the fluorescence yield at the J-step (2 ms). Upon prolongation of the anaerobic treatment (> 60 min), the O-J-I-P fluorescence rise was eventually transformed to an O-J (J = P) rise. A similar transformation was observed when pea leaves were treated with DCMU or sodium dithionite. Anaerobiosis resulted in a 10–20% reduction in the maximum quantum yield of the primary photochemistry of Photosystem II, as measured by the ratio of the maximal values of variable and total fluorescence (F_V/F_M). When the leaves were returned to the air in the dark, the shape of the fluorescence transient showed a time-dependent recovery from the anaerobiosis-induced change. The original O-J-I-P shape could also be restored by illuminating the anaerobically treated samples with far-red light but not with blue or white light. Osmotically broken chloroplasts displayed under anaerobic conditions fluorescence transients similar to those observed in anaerobically treated leaves, but only when they were incubated in a medium comprising reduced pyridine nucleotides (NADPH or NADH). As in intact leaves, illumination of the anaerobically treated chloroplasts by far-red light restored the original O-J-I-P transient, although only in the presence of methyl viologen. The results provide additional evidence for the existence of a chlororespiratory pathway in higher plant cells. Furthermore, they suggest that the J-level of the fluorescence transient is strongly determined by the redox state of the electron carriers at the PS II acceptor side.     

Differential response of Brassica juncea cultivars to Al; consequences for chlorophyll a fluorescence,antioxidants and psb A gene

The present study was undertaken to identify the aluminum (Al) tolerant cultivar of Brassica juncea. We examined the changes in antioxidant enzymes, proline level, chlorophyll a fluorescence and psb A gene expression at vegetative and reproductive growth stages of B. juncea cultivars (Bio-902, CS-14, Pusa-Tarak and Laxmi). The selected cultivars were exposed to soil (pH 5.2) supplemented with Al (0, 50, 100 and 150?mg?kg^?1). We observed the lowest decline in photosynthetic efficiency (ΔF/Fm′), ETR, PPFD values and psb A expression to Al stress in the cultivar Bio-902 (tolerant cultivar) followed by CS-14 and Pusa-Tarak whereas the highest decline was observed in the cultivar Laxmi (sensitive cultivar). The improved performance of the cultivar Bio-902 under Al stress was accompanied by an increase in proline level, CAT and APX activities but without any increase in SOD activity. However, significant increase in SOD activity was observed in Laxmi.     

三峡库区古夫河着生藻类叶绿素a的时空分布特征及其影响因素

古夫河系长江三峡水库湖北省境内香溪河的一条支流,发源于神农架林区并最终汇入三峡水库香溪河库湾,是流域生态学研究的热点水体。本文对古夫河2010年9月至2011年8月干流及主要支流21个样点的着生藻类叶绿素a和14项环境因子进行了调查,采用多元统计方法对调查数据进行了方差分析、聚类分析、偏相关分析和逐步回归分析,研究了着生藻类叶绿素a的时空分布特征及其主要环境影响因子。结果表明：叶绿素a含量范围为0.07—145.96 mg/m2,平均值为11.63 mg/m2。不同样点的叶绿素a含量差异显著,其中古夫河干流上游低于下游,支流竹园河上游高于下游,表现为人为影响大的区域高于人为影响小的区域;不同季节着生藻类叶绿素a含量差异显著,表现出冬春季高、夏秋季低的趋势。古夫河着生藻类叶绿素a与总磷和硬度呈极显著正相关,与电导率、氨氮和总氮呈显著正相关,而与流速呈极显著负相关;水体总磷是古夫河流域着生藻类生长的第一限制因子,流速对着生藻类的生长具有显著抑制作用;古夫河着生藻类生物量空间格局可能由其生境尺度（营养盐）和流域尺度（硬度和电导率）的特征决定,而古夫河着生藻类生物量时间变化主要受水动力（流速）的影响。     

Comparison of chlorophyll fluorescence emission characteristics of wheat leaf tissue and isolated thylakoids as a function of excitation wavelength

Abstract. Chlorophyll fluorescence emission spectra and the kinetics of 685 mm fluorescence emission from wheat leaf tissue and thylakoids isolated from such tissue were examined as a function of excitation wavelength. A considerable enhancement of fluorescence emission above 700 nm relative to that at 685 nm was observed from leaf tissue when it was excited with 550 nm rather than 450 nm radiation. Such excitation wavelength dependent changes in the emission spectrum occurred over an excitation spectral range of 440–660 nm and appeared to be directly related to the total quantity of radiation absorbed at a given excitation wavelength. Experiments with isolated thylakoid preparations demonstrated that changes in the fluorescence emission spectrum of the leaf were attributable to the optical properties of the leaf and were not due to the intrinsic characteristies of the thylakoid photochemical apparatus. This was not the case for the observed excitation wavelength dependent changes in the 685 nm fluorescence induction curve obtained from leaf tissue infiltrated with DCMU. Excitation wavelength dependent changes in the ratio of the variable to maximal fluorescence emission and the shape of the variable fluorescence induction were observed for leaf tissue. Isolated thylakoid studies showed that such changes in the leaf fluorescence kinetics were representative of the way in which the photochemical apparatus in vivo was processing the absorbed radiation at the different excitation wavelengths. The results are considered in the context of the use of fluorescence emission characteristics of leaves as non-destructive probes of the photochemical apparatus in vivo.     

Biological hydrogen production by the algal biomass Chlorella vulgaris MSU 01 strain isolated from pond sediment

Chlorella vulgaris MSU 01 strain isolated from the sediment of the pond is able to produce molecular hydrogen in a clean way. To relate the dynamic coupling between the cultural conditions and biological responses, an original lab scale set up has been developed for hydrogen production. Different sources like mannitol, glucose, alanine, citric acid, aspartic acid, l-alanine, l-cysteine, sodium succinate and sodium pyruvate were used for algal media optimization. Corn stalk, from 1 to 5 g/L was tested for the effective algal growth and hydrogen production. The cell concentration of 1.6-19 g/L dry cell weight (DCW) was found at the 10th day. The kinetic parameters involved in the hydrogen production at 4 g/L corn stalk using the algal inoculum (50 mL) in the bioreactor volume (500 mL) was found to be with the hydrogen production potential (P_s) of 7.784 mL and production yield of (P_r) 5.534 mL respectively. The growth profile of the algal biomass at the above mentioned condition expressed the logistic model with R² 0.9988. The final pH of the broth was increased from 7.0 to 8.5-8.7. The anaerobic fermentation by C. vulgaris MSU 01 strain involved in the conversion process of complex carbon source has increased the H₂ evolution rate and higher butyrate concentration in the fermentate.